WB实验中需要各种缓冲液，试剂的配置配方也是多种多样，各项配比虽相差不多，但也是让人看花了眼。Western免疫印迹（Western Blot）是将蛋白质转移到膜上，然后利用抗体进行检测的方法。对已知表达蛋白，可用相应抗体作为一抗进行检测，对新基因的表达产物，可通过融合部分的抗体检测。
与Southern或Northern杂交方法类似，但Western Blot采用的是聚丙烯酰胺凝胶电泳，被检测物是蛋白质，“探针”是抗体，“显色”用标记的二抗。
经过PAGE分离的蛋白质样品，转移到固相载体（例如硝酸纤维素薄膜）上，固相载体以非共价键形式吸附蛋白质，且能保持电泳分离的多肽类型及其生物学活性不变。以固相载体上的蛋白质或多肽作为抗原，与对应的抗体起免疫反应，再与酶或同位素标记的第二抗体起反应，经过底物显色或放射自显影以检测电泳分离的特异性目的基因表达的蛋白成分。该技术也广泛应用于检测蛋白水平的表达。
一般的WB检测过程中，都会有封闭、一抗、二抗和底物显色这四道工序要“加工”，其中底物显色这最后一步是最关键的。标记方式有生物素标记、地高辛标记、各种酶标记等等，酶标的底物又有各种生色底物、化学发光法底物和荧光底物可供选择。
最熟悉的底物应该是3'3'二氨基联苯胺DAB，DAB可以和HRP作用形成褐色不溶性产物，灵敏度高，特异性好，缺点就是需要现配现用，有一定的毒性，显色后光照数小时会
褪色，不能永久保存，需要拍照记录。
HRP化学发光反应底物经过不断研究开发而有了更显著的改进。Luminol是经典的HRP化学发光底物。化学发光底物主要考虑的是：发光信号强弱——发光信号当然越强越好，强发光信号持续的时间——也是越长越好，还有就是背景低灵敏度高。化学发光法由于仅在酶和底物都存在的时候才会发光，不同于生色反应般形成不溶的产物于膜上累积，所以特别适合同一张膜对不同的目标蛋白分别进行多次检测。目前最高检测灵敏度已经达到低飞克（Femto）级别而成为安全且灵敏度最高的WB检测方法。
南京巴傲得生物科技有限公司 ( Biogot technology, co, Ltd），是专一服务于生命科学研究的专业技术型企业。是一大批志同道合的资深专业人士共同创办的具有现代化经营管理水平的生物高科技企业，从事生物技术产品的开发及销售，致力于将巴傲得生物打造成中国乃至世界一流的生物技术产品供应商。巴傲得生物长期备有Bioworld抗体现货8000多种抗体可以在一周左右送到客户的手里。精湛品质、合理价格、专业服务，是我们为您服务的宗旨。在细胞信号通路、免疫学、蛋白组学上拥有显著优势，其抗体产品、生长因子等为生命科学科研工作者提供了极大的便利。客户的满意、微笑和信任是对我们最大的鼓舞与激
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Various buffers are needed in WB experiments, and the formulations of reagents are also varied. Although the ratios are similar, they are also eye-catching. Western Blot is a method of transferring protein to membrane and then detecting it with antibody. For the known expressed protein, the corresponding antibody can be used as the primary antibody for detection, and for the expression product of the new gene, the antibody of the fusion part can be used for detection.
Similar to Southern or Northern hybridization, Western Blot uses polyacrylamide gel electrophoresis, the detected substance is protein, \is antibody, and \
The protein sample separated by PAGE is transferred to a solid carrier (such as nitrocellulose membrane), and the solid carrier adsorbs protein in the form of noncovalent bond, and can keep the type of polypeptide separated by electrophoresis and its biological activity unchanged. Protein or polypeptide on solid carrier is used as antigen, which reacts with corresponding antibody, and then reacts with second antibody labeled by enzyme or isotope, and the protein component expressed by specific target gene separated by electrophoresis is detected by substrate coloration or autoradiography. This technique is also widely used to detect protein expression.
In the general WB detection process, there are four steps to be \namely, blocking, primary antibody, secondary antibody and substrate color development, and the last step of substrate color development is the most critical.
Labeling methods include biotin labeling, digoxigenin labeling, various enzyme labeling, etc. The substrates of enzyme labeling can be selected from various chromogenic substrates, chemiluminescence substrates and fluorescent substrates.
The most familiar substrate should be 3'3' diaminobenzidine DAB, which can react with HRP to form brown insoluble product, with high sensitivity and good specificity. The disadvantage is that it needs to be prepared and used now, and it has certain toxicity. After color development, it will fade after illumination for several hours, and it cannot be stored permanently, so it needs to be photographed and recorded.
After continuous research and development, the substrate of HRP chemiluminescence reaction has been significantly improved. Luminol is a classical substrate of HRP chemiluminescence. Chemiluminescence substrates mainly consider the intensity of luminescence signal-the stronger the luminescence signal, the better, and the longer the duration of the strong luminescence signal, the better, and the low background and high sensitivity. Chemiluminescence method is especially suitable for multiple detections of different target proteins on the same membrane because it only emits light when both enzymes and substrates exist, and forms insoluble products to accumulate on the membrane unlike chromogenic reaction. At present, the highest detection sensitivity has reached the low femtolevel, making it a safe and highly sensitive WB detection method.
Bioworld technology co., Ltd manufactures and supplies antibodies for worldwide distribution. We provide antibodies in bulk quantity and OEM basic
services. The experienced scientific and technical professionals make Bioworld Technology a highly innovative, first-class biotechnological company on both manufacturing and marketing. Bioworld strives for a national leader and the world-class company in manufacturing biological products for industry, health, and research. We look forward to serving your research needs and becoming your long-term research partner.
WB试剂（含二抗）
